HMEC-1 cells were purchased from ATCC (CRL-3243™) and were grown in MCDB131 base medium in presence of Epidermal Growth Factor (10 ng/mL), Hydrocortisone (1 μg/mL), 10 mM Glutamine and a final concentration of 10% Fetal Bovine Serum (FBS). THP-1 cells were culuted in RPMI-1640 medium containing 10% FBS and 1% Pen-Strep. The cells were grown in Cellstar® Filter cap 75 cm2 flasks in humidified incubators at 37 °C and 5% CO2. Respective media was renewed every 2 days, and cells were split into a new passage at 85% confluence.
Crl 3243
Manufactured by American Type Culture Collection
Sourced in United Kingdom, United States
The CRL-3243 is a piece of laboratory equipment used for cell culture applications. It is designed to provide a controlled environment for the growth and maintenance of cells in vitro. The core function of this product is to regulate temperature, humidity, and gas composition to support optimal cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Panc 1, by Merck Group (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Dmem with high glucose, by Merck Group (1 mentions)
Mcdb 131 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Hmec 1, by American Type Culture Collection (1 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
Mcdb131, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Welgene (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Ccl 10, by American Type Culture Collection (1 mentions)
6 protocols using crl 3243
1
HMEC-1 and THP-1 Cell Culture Protocol
2
Expansion of Cell Lines for Cancer Research
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The human pancreatic adenocarcinoma cell line PANC-1 (Sigma–Aldrich, Merck, United Kingdom) was expanded in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Sigma–Aldrich, Merck, United Kingdom) supplemented with 10% fetal bovine serum (FBS, Fisher Scientific, United Kingdom), 1% penicillin/streptomycin (Fisher Scientific, United Kingdom), and 2 mM L -glutamine (Sigma–Aldrich, Merck, United Kingdom) in a humidified incubator at 37°C with 5% CO2.
The HMEC line CRL-3243 (ATCC, United Kingdom) was expanded in MCDB 131 medium (GIBCO, Thermo Fisher, United Kingdom), supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mML -glutamine, 10 ng/ml epidermal growth factor (SIGMA–Aldrich, Merck, United Kingdom), and 1 μg/ml hydrocortisone (SIGMA–Aldrich, Merck, United Kingdom) in a humidified incubator at 37°C with 5% CO2.
The immortalized human pancreatic stellate cells (PS-1) were expanded in DMEM/F12 medium (GIBCO, Thermo Fisher, United Kingdom) supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mML -glutamine in a humidified incubator at 37°C with 5% CO2.
All cells were passaged regularly on reaching 80–90% confluency with TrypLE (GIBCO, Thermo Fisher, United Kingdom) until the required cell densities were obtained.
The HMEC line CRL-3243 (ATCC, United Kingdom) was expanded in MCDB 131 medium (GIBCO, Thermo Fisher, United Kingdom), supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM
The immortalized human pancreatic stellate cells (PS-1) were expanded in DMEM/F12 medium (GIBCO, Thermo Fisher, United Kingdom) supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mM
All cells were passaged regularly on reaching 80–90% confluency with TrypLE (GIBCO, Thermo Fisher, United Kingdom) until the required cell densities were obtained.
3
Cell Culture Protocols for Various Cell Lines
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Human epidermal keratinocytes (HEKn, Gibco, Waltham, MA, USA) were maintained in EpiLife medium (Gibco) supplemented with Human Keratinocyte Growth Supplement (HKGS, Gibco) at 37 °C in the presence of 5% CO2. THP-1 human monocytes were maintained in RPMI 1640 medium (WELGENE Inc., Gyeongsan, Korea) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (Gibco) at 37 °C under 5% CO2. Mouse mast cell line MC/9 was maintained in Dulbecco’s Modified Eagle Medium (DMEM), containing 10% T-Cell Culture Supplement with ConA (T-STIM, Corning, NY, USA), 10% FBS, 0.05 mM 2-mercaptoethanol (Sigma Aldrich), and 1% penicillin/streptomycin, at 37 °C, under 5% CO2. Human endothelial microvascular cell lines (HMEC-1, CRL-3243™, ATCC® Manassas, VA, USA) were maintained in MCDB 131 (Gibco), containing 10 ng/mL EGF (Invitrogen, Carlsbad, CA, USA), 1 µg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 mM glutamine (Gibco), and 10% FBS at 37 °C in the presence of 5% CO2.
4
Dengue Virus Type 2 Propagation Protocol
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Dengue virus type 2 (DENV-2, New Guinea strain), originally donated by María Elena Peñaranda and Eva Harris (University of California, Berkeley) was amplified in C6/36 HT cells (Aedes albopictus-ATCC®® CRL-1660™) and titrated in BHK-21 cells (hamster kidney cells, ATCC®® CCL-10™). HMEC-1 cells (human dermal microvascular endothelial cells, ATCC®® CRL-3243™) were maintained in basal growth medium (10% FBS RPMI supplemented with 10 mM L-glutamine, 100 units/mL penicillin/streptomycin (P/S), 10 ng/mL epidermal growth factor (hEGF), 1 µg/mL hydrocortisone) and incubated at 37 °C in a 5% CO2, humidified atmosphere. BHK-21 cells were maintained in 10% FBS DMEM supplemented with 100 U/mL penicillin/streptomycin, and 10 mM L-Glutamine incubated at 37 °C and 5% CO2 and C6/36 HT cells with 10% FBS L-15 medium and 100 U/mL P/S at 34 °C and 5% CO2.
5
In Vitro Diabetic Retinal Cell Culture
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A human retinal pericyte (HRP) line was stabilized in our laboratory [14] (link). Human microvascular endothelial cells (HMECs) were purchased from ATCC (Cat#CRL-3243, RRID:CVCL_0307), and the human Müller cell line Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1) (RRID:CVCL_0433) obtained from the UCLB licensing portal XIP (London, UK) [15] . HRP and MIO-M1 cells were cultured in DMEM+10%FCS; HMEC in EBM-2 growth medium (Lonza) for expansion and DMEM+10%FCS for experiments.
Cells were cultured for 8 days in physiological conditions (NG, 5.6 mmol/l D-glucose), or intHG (48hr high glucose, 28 mmol/l /48hr NG, twice), to better mimic the diabetic microenvironment, since we had previously demonstrated that human pericytes are more affected by intermittent high glucose conditions [16] (link). 50 µmol/l thiamine (T) and 100 µmol/l fenofibric acid (FA) (concentrations chosen on the basis of previous reports [7, (link)10] (link)) were added during the whole 8-day incubation. Hypoxic conditions (hypo) were obtained by keeping cultures in a 5%CO 2 / 94%N 2 /1%O 2 gas mixture for the last 48 hrs.
Reagents for cell cultures were purchased from Sigma-Aldrich, unless otherwise stated.
Cells were cultured for 8 days in physiological conditions (NG, 5.6 mmol/l D-glucose), or intHG (48hr high glucose, 28 mmol/l /48hr NG, twice), to better mimic the diabetic microenvironment, since we had previously demonstrated that human pericytes are more affected by intermittent high glucose conditions [16] (link). 50 µmol/l thiamine (T) and 100 µmol/l fenofibric acid (FA) (concentrations chosen on the basis of previous reports [7, (link)10] (link)) were added during the whole 8-day incubation. Hypoxic conditions (hypo) were obtained by keeping cultures in a 5%CO 2 / 94%N 2 /1%O 2 gas mixture for the last 48 hrs.
Reagents for cell cultures were purchased from Sigma-Aldrich, unless otherwise stated.
6
CRISPR/Cas9 Transfection in Fibroblasts and Endothelial Cells
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